Hammond John, Reinsel Michael, Grinstead Samuel, Lockhart Ben, Jordan Ramon, Mollov Dimitre
Floral and Nursery Plants Research Unit, United States National Arboretum, United States Department of Agriculture-Agricultural Research Service, Beltsville, MD, United States.
National Germplasm Resources Laboratory, Beltsville Agricultural Research Center, United States Department of Agriculture-Agricultural Research Service, Beltsville, MD, United States.
Front Microbiol. 2020 Dec 21;11:612936. doi: 10.3389/fmicb.2020.612936. eCollection 2020.
Multiple carlaviruses infect various ornamental plants, often having limited host ranges and causing minor symptoms, yet often reducing yield or quality. In this study we have identified a mixed infection of butterbur mosaic virus (ButMV) and helenium virus S (HelVS) from a plant of veronica ( sp.) showing foliar mosaic and distortion. Carlavirus-like particles were observed by transmission electron microscopy (TEM), and RNA from partially purified virions was amplified by random RT-PCR, yielding clones of 439-1,385 bp. Two partially overlapping clones including coat protein (CP) sequence, and two of four partial replicase clones, were closely related to ButMV-J (AB517596), previously reported only from butterbur () in Japan. Two other partial replicase clones showed lower identity to multiple carlaviruses. Generic primers which amplify the 3'-terminal region of multiple carlaviruses yielded clones of three distinct sequences: (1) with 98% nt identity to HelVS; (2) ButMV-A, showing 82% nt identity to ButMV-J; and (3) ButMV-B, with 78% nt identity to each of ButMV-J and ButMV-A. Further amplification of upstream fragments revealed that ButMV-B had an internal deletion in TGB1, confirmed using isolate-specific primers. Near-complete genomes of both ButMV-A and ButMV-B were obtained from next-generation sequencing (NGS), confirming the deletion within ButMV-B, which is presumably maintained through complementation by ButMV-A. HelVS was previously reported only from hybrids and . A near-complete HelVS genome was obtained for the first time by NGS from the same sample. Additional hybrids infected with HelVS were identified by TEM and RT-PCR, including cv. 'Sunny Border Blue' which was also subjected to NGS. This resulted in assembly of an 8,615 nt near-complete HelVS genome, with high identity to that from the mixed infection. The predicted CP sequence has 96% amino acid (aa) identity to HelVS from helenium (Q00556). Other ORFs show a maximum of 54% (TGB3) to 68% (NABP) aa identity to the equivalent ORFs of other carlaviruses. These results demonstrate for the first time maintenance by complementation of a carlavirus isolate with a major deletion in an essential gene, and confirm that HelVS is a distinct species in the genus .
多种香石竹潜隐病毒感染各种观赏植物，通常寄主范围有限，症状较轻，但常导致产量下降或品质降低。在本研究中，我们从一株出现叶片花叶和扭曲症状的婆婆纳属（Veronica sp.）植物中鉴定出了蜂斗菜花叶病毒（ButMV）和堆心菊病毒S（HelVS）的混合感染。通过透射电子显微镜（TEM）观察到了类香石竹潜隐病毒粒子，并用随机RT-PCR扩增了部分纯化病毒粒子的RNA，得到了439 - 1385 bp的克隆。两个包含外壳蛋白（CP）序列的部分重叠克隆以及四个部分复制酶克隆中的两个，与之前仅在日本的蜂斗菜（Petasites japonicus）中报道过的ButMV-J（AB517596）密切相关。另外两个部分复制酶克隆与多种香石竹潜隐病毒的同源性较低。扩增多种香石竹潜隐病毒3'-末端区域的通用引物产生了三个不同序列的克隆：（1）与HelVS的核苷酸（nt）同源性为98%；（2）ButMV-A，与ButMV-J的nt同源性为82%；（3）ButMV-B，与ButMV-J和ButMV-A的nt同源性均为78%。上游片段的进一步扩增表明ButMV-B在TGB1中有内部缺失，使用分离物特异性引物得以证实。通过二代测序（NGS）获得了ButMV-A和ButMV-B的近全长基因组，证实了ButMV-B中的缺失，推测其通过ButMV-A的互补得以维持。HelVS之前仅在堆心菊属（Helenium）杂种和堆心菊（H. autumnale）中报道过。通过NGS首次从同一样本中获得了近全长的HelVS基因组。通过TEM和RT-PCR鉴定出了另外一些感染HelVS的堆心菊属杂种，包括品种‘Sunny Border Blue’，该品种也进行了NGS分析。这导致组装出了一个8615 nt的近全长HelVS基因组，与混合感染中的HelVS基因组具有高度同源性。预测的CP序列与来自堆心菊（Q00556）的HelVS的氨基酸（aa）同源性为96%。其他开放阅读框（ORF）与其他香石竹潜隐病毒的等效ORF的aa同源性最高为54%（TGB3）至68%（NABP）。这些结果首次证明了一种在必需基因中存在主要缺失的香石竹潜隐病毒分离物通过互补得以维持，并证实HelVS是该属中的一个独特种。
